Li Shiyu, Zeng Shuangshuang, Wen Chenyu, Zhang Zhen, Hjort Klas, Zhang Shi-Li
Department of Electrical Engineering, Division of Solid-State Electronics, Uppsala University, SE-751 03 Uppsala, Sweden.
Department of Material Science and Engineering, Division of Microsystem Technology, Uppsala University, SE-751 21 Uppsala, Sweden.
ACS Sens. 2022 May 27;7(5):1476-1483. doi: 10.1021/acssensors.2c00216. Epub 2022 May 10.
Integration of motor enzymes with biological nanopores has enabled commercial DNA sequencing technology; yet studies of the similar principle applying to solid-state nanopores are limited. Here, we demonstrate the real-life monitoring of phi29 DNA polymerase (DNAP) docking onto truncated-pyramidal nanopore (TPP) arrays through both electrical and optical readout. To achieve effective docking, atomic layer deposition of hafnium oxide is employed to reduce the narrowest pore opening size of original silicon (Si) TPPs to sub-10 nm. On a single TPP with pore opening size comparable to DNAP, ionic current measurements show that a polymerase-DNA complex can temporally dock onto the TPP with a certain docking orientation, while the majority become translocation events. On 5-by-5 TPP arrays, a label-free optical detection method using Ca sensitive dye, are employed to detect the docking dynamics of DNAP. The results show that this label-free detection strategy is capable of accessing the docking events of DNAP on TPP arrays. Finally, we examine the activity of docked DNAP by performing on-site rolling circle amplification to synthesize single-stranded DNA (ssDNA), which serves as a proof-of-concept demonstration of utilizing this docking scheme for emerging nanopore sensing applications.
将运动酶与生物纳米孔相结合已促成了商业化的DNA测序技术；然而，将类似原理应用于固态纳米孔的研究却很有限。在此，我们展示了通过电学和光学读出对phi29 DNA聚合酶（DNAP）对接至截顶金字塔形纳米孔（TPP）阵列进行实际监测。为实现有效对接，采用氧化铪的原子层沉积将原始硅（Si）TPP的最窄孔径减小至10纳米以下。在孔径与DNAP相当的单个TPP上，离子电流测量表明，聚合酶-DNA复合物能够以特定的对接方向暂时对接至TPP，而大多数则成为易位事件。在5×5的TPP阵列上，采用一种使用钙敏感染料的无标记光学检测方法来检测DNAP的对接动力学。结果表明，这种无标记检测策略能够检测到DNAP在TPP阵列上的对接事件。最后，我们通过进行原位滚环扩增以合成单链DNA（ssDNA）来检测对接的DNAP的活性，这作为利用这种对接方案用于新兴纳米孔传感应用的概念验证演示。
